Epstein-Barr virus (EBV) infection in AIDS patients is associated with both B-cell lymphomas and oral hairy leukoplakia. The EBV immediate-early (IE) proteins, BZLF1 and BRLF1, mediate the switch between the latent and lytic forms of EBV infection. In addition, the IE proteins have a profound effect on the host cell environment. In this competitive grant renewal, we propose to continue our long-term studies on the role of the two EBV IE proteins in EBV-associated diseases. We hypothesize that the two EBV IE proteins are not only required to induce the lytic form of EBV infection, but may also help to support certain EBV-associated malignancies through a paracrine mechanism. We have recently discovered that BZLF1 preferentially binds to, and activates, the BRLF1 IE promoter only after it has been methylated over a CpG motif contained within a BZLF1 binding site. Thus, BZLF1 appears to be uniquely suited for activating the methylated form of the viral genome. We have also recently identified a new viral transactivator protein, BRRF1, and shown that BRRF1 collaborates with BRLF1 to induce lytic EBV infection. In addition, recent data from our laboratory suggest that paracrine factors released from lytically infected cells are required for the development of EBV-associated lymphomas in vivo. Our specific aims are as follows. In Specific Aim 1, we will compare and contrast how epigenetic modifications of the two IE viral promoters (including DNA methylation and histone acetylation) affect the ability of BZLF1 versus BRLF1 to disrupt viral latency, and determine whether the novel ability of BZLF1 to preferentially activate the methylated form of the BRLF1 IE promoter is important for BZLF1 function. In Specific Aim 2, we will further investigate the mechanisms by which BRLF1 and BRRF1 both individually transactivate the BZLF1 IE promoter in EBV-negative cells, and collaborate to induce lytic infection in EBV-positive cells. In Specific Aim 3, we will use wild-type, as well as BZLF1 - and BRLF1-knock-out viruses, to infect epithelial cells and determine if IE proteins are important for viral pathogenesis or transformation in these cells. The proposed studies should yield important insight into the multiple roles that lytic EBV proteins play in EBV-associated diseases.